APPLICANT'S DESCRIPTION: The goal of this proposal is to evaluate the transplantation of positively selected autologous hematopoietic stem cells (HSC) in pediatric patients with acute lymphoblastic leukemia (ALL). Previous results with autologous HSC transplantation (HSCT) for ALL have reported an unacceptable high relapse rates possibly due to leukemia cells contaminating the transplanted HSC. HSC will be positively selected by fluorescence activated cell sorting (FACS) based upon immunophenotypic differences between HSC (CD34+, CD38-, yc-) and ALL cells (CD34+, CD38+, yc+). Positively selected HSC will be evaluated 1) for their hematopoietic potential and 2) the presence of residual leukemia cells by PCR analysis for leukemia specific clonotypic TCR-6 rearrangements. PCR analysis of the clonospecific TCR-6 rearrangement permits an independent assessment of the purity of the isolated HSC (Specific Aim 1). All patients will be prepared for HSCT with total body irradiation and VP-16, and their rate of lymphohematopoietic reconstitution determined following the transplantation of the positively selected HSC (Specific Aim 2). After the demonstration that positively selected HSC can engraft in a clinically relevant time frame, an aliquot of the isolated HSC will be transduced with a glucocerebrosidase (GC) containing lentivirus based vector prior to cryopreservation. Both the transduced and non-transduced HSC will then be transplanted. Lentivirus based vectors will be used since our previous work has shown that murine leukemia based vectors poorly transduced quiescent HSC. Using inverse PCR techniques the clonality and the multi- lineage progeny of the transduced HSC will be longitudinally assessed. If patients relapse, their relapsed leukemia cells will be isolated and assess for the presence of the transduced vector to determine if the transplanted HSC contributed to relapse (Specific Aim 3). Overall this grant is an attempt to demonstrate that the transplantation of positively selected HSC (Cb34+, CD38-, yc-) can successfully lymphohematopoietic reconstitute pediatric ALL patients and that lentivirus vectors can transduce pluripotent HSC.